Biological tissue, or simply tissue, as used herein includes a plurality of cells or any cellular material that carry out a biological function. Any plant or animal tissue is included. Animal organs or whole animals, such as Planaria, are included. The cells are not necessarily identical, but are preferably of the same origin.
The primary storage mechanism employed in this invention uses tissue storage temperatures above zero degrees Centigrade, (i.e., above the freezing point of water at atmospheric pressure) in the presence of a gaseous chemical agent. The tissue remains viable in long or short term storage, essentially hibernating with significantly reduced biological function. The process enables the tissue to be later recovered to a viable state, that is, to a state with normal biological function.
The present invention can also be employed for biological tissue storage in a specific range of below freezing temperatures using this same gaseous chemical agent. These embodiments also maintain a viable tissue that is capable of restoration of normal tissue function when recovered to above freezing temperatures.
Conventional processes that seek to preserve tissue at low temperatures include near-freezing storage; conventional vitrification, and conventional cryopreservation. None teaches the process of the present invention and all suffer from overall inadequate performance in viable storage and recovery to a state of normal function.
Lowering the temperature as a preservative of biological tissue is known in the art. See for example U.S. Pat. No. 5,791,151 which uses a near-freezing temperature in an oxygen environment. However, sustaining viability of tissue and recovering the tissue to normal function with the teachings in the prior art is problematic.
Prior art processes typically use liquid polar organic compounds in solution to perfuse the biological tissue. These conventional processes are not reversible in that they cannot be used to restore tissue to life, although occasional exceptions are observed in nature that involve, for example, vitrifying polyols (i.e., insects, amphibians) or thermal hysteresis proteins (insects, fish). See, Fletcher G L, Hew C L and Davies P L, Antifreeze proteins of teleost fishes. Annu Rev Physiol, 63 (2001) 359-590; Graham L A, Liou Y C, Walker V K and Davies P L, Hyperactive antifreeze protein from beetles. Nature, 388 (1997) 727-728. The present method does not employ a liquid, but rather employs a specific gaseous chemical agent in a specific process that enhances the viability of biological tissue in short and long-term storage and enhances recovery of that tissue when required.
Near-freezing storage seeks to preserve organs by lowering their temperature near to the freezing point of water. See, e.g., U.S. Pat. No. 7,029,839. Near-freezing storage involves perfusing the tissue with an aqueous solution containing protectants that depress the freezing point of the solution, so that the tissue may be stored at low temperature with aqueous fluids in the cells in a liquid state. Examples of liquid polar organic compounds used as conventional protectants are dimethyl sulfoxide, glygerol, ethylene glycol, and propylene glycol. Conventional protectants can function by binding water through a combination of hydrophilic and hydrophobic interactions at different points on the molecule.
Conventional protectants can present problems when used on larger pieces of tissue; such problems are generally attributed to the nonuniform distribution of the protectants within the tissue. Conventional protectants typically diffuse slowly and pass through cell membranes and the blood-brain barrier poorly or not at all. Furthermore, large quantities of protectants may be required.
Typically, conventional protectants bind with about two moles of water per mole of protectant. When used in the required quantities to bind water conventional protectants may be toxic to cells. The near-freezing storage process is slow and requires that high concentrations of potentially harmful protectant chemicals be introduced to and removed from the tissue.
In general, preserving biological tissue by lowering its temperature below freezing is destructive of cellular tissue when crystalline ice forms within the cells (intracellular) and around cells (extracellular) as the liquid water within the biological tissue transitions to the solid phase (ice). The mechanism of freezing damage in living tissue is principally due to two processes.
The first process causing freezing damage involves the formation of ice in the intercellular spaces. The vapor pressure of the ice is lower than the vapor pressure of the solute water in the surrounding cells and as heat is removed at the freezing point of the solutions, the ice crystals grow between the cells, extracting water from them. As the ice crystals grow, the volume of the cells shrinks, and the cells are crushed between the ice crystals.
The second process causing freezing damage involves the concentration of solutes inside the water remaining in the cells as the cells shrink. The increased concentration of solutes increases the intracellular ionic strength and interferes with the organization of proteins and other intercellular structures. Eventually, the solute concentration inside the cells reaches the eutectic and freezes. The final state of the frozen tissue is pure ice in the extracellular spaces, and a mixture of concentrated cellular components in ice and bound water inside the cells.
Most lesions in tissue occur during re-warming and reperfusion of cryopreserved biological tissues, such as organs; the process of its development is time-consuming. Changes include condensation of chromatin, large lipid droplets, and partly disrupted plasma membrane; these changes may be seen on electron microscopy (which may be a consequence of the osmotic excursions incurred during a freeze-thaw cycle; leakage of mitochondrial matrix can trigger apoptosis as well).
Damage to biological tissue by freezing is caused, besides temperature stress owing to decrease in temperature itself, by the following processes: irreversible change of biological membrane by dehydration from the cells and surface of the membranes caused by the freezing process; destruction by loss in selective permeability; and, physical deformation and death of the cell. Light microscopy does not show early freezing damage to the cells. The present invention avoids such damage.
Conventional vitrification involves the use of a conventional cryoprotectant solution and cryogenic temperatures. See, e.g., U.S. Pat. No. 4,559,298. A concentrated aqueous cryoprotectant solution can permit solidification without the formation of ice crystals. That is, vitrification can involve inducing the transition of an aqueous liquid to an amorphous solid phase in both the intracellular and the extracellular spaces of tissue by cooling to a cryogenic temperature with the use of a conventional cryoprotectant, such as glycerol. However, vitrification requires the impregnation of biological tissues with high concentrations of toxic cryoprotective chemicals that promote the vitreous state.
Although vitrification can avoid ice formation, alternative potential mechanisms of injury associated with the amorphous state have been identified. Devitrification (ice formation in biological tissues during re-warming) is a major obstacle to successful organ vitrification and subsequent recovery. Vitrification has failed to successfully preserve and return to a viable state mammalian internal organs.
Conventional cryopreservation can involve the use of liquid cryoprotectant solutions to prevent intracellular ice crystal formation, while allowing ice crystals to form in extracellular areas. In addition to using potentially toxic protectant chemicals, conventional vitrification and conventional cryopreservation techniques can cause cells to undergo volume changes during vitrification or freezing, which results in mechanical stresses sufficient to cause cracking and cell destruction.
The use of xenon in cryopreservation was discussed by P. V. Shcherbakov and V.1. Telpuhov. See, P.V. Shcherbakov and V.1. Telpuhov, Chemistry and Life, v.8 (2006) pp. 34-39 (in Russian). Additionally, Russian patent RU2268590 to Shcherbakov, et al. (published Jan. 27, 2006 with English language Abstract) discusses saturating tissue with a mixture of xenon, krypton, and argon, forcing water out of the tissue with this mixture of noble gases under pressure while cooling to −43° C., and decreasing the pressure to ambient pressure and continuing to cool to −196° C. The present invention does not employ cryopreservation temperatures as taught by Shcherbakov. The pressure of the noble gas mixture presented in the Shcherbakov publications does not allow for sufficient water to be bound in the cells to allow for rehydration sufficient for metabolism to restart. The present invention utilizes a specific gas mixture and a specific concentration not taught in the Shcherbakov publications. Further, the Shcherbakov publications do not present a method suitable for viably storing tissue capable of recovery to a viable state as enabled in the present invention.